The ability of neutrophils kill ingested microorganisms is essential for host defense against microbial infection. Our long term goal is to identify the endogenous antibiotic peptides of human neutrophils. The present application has six specific aims. l) To purify """"""""granulins"""""""" from human neutrophils, test their antimicrobial, growth-regulatory and cytotoxic properties, and determine their subcellular localization and their mode of release. 2) To determine if human neutrophils contain antimicrobial members of the """"""""four-disulphide-core family"""""""" of basic proteins. 3) To establish the prevalence of neutrophil defensin deficiency in inbred strains of mice and to purify and identify the non- defensin antimicrobial proteins of murine neutrophils. 4) To examine selected properties of human defensin HNP-4 relevant to its antimicrobial, cytotoxic and corticostatic functions. 5) To express human defensins in otherwise defensin-deficient murine phagocytes and to determine the consequences for antimicrobial activity against H. capsulatum and C. albicans. 6) To characterize the binding of human defensins to bacteria and human mononuclear cells, and to study the uptake of exogenous defensins by human monocytes and macrophages and determine its consequences for antimicrobial activity against H. capsulatum and C. albicans. The methods used in Specific Aims 1-4 involve cellular and subcellular fractionations, analytical and preparative protein chemistry and various microbiological assays.
Specific Aim 5 combines molecular biology and microbiology and will use recently constructed murine cell lines that express and process the human defensin HNP-1.
Specific Aim 6 will combine immunoelectron microscopy with measurements of binding and uptake of defensins and quantitative measurements of macrophage-mediated antimicrobial activity. Overall, these studies will define the role of defensins and other cysteine-rich antimicrobial peptides (granulins and four-disulphide-core congeners) in the antimicrobial activity of human neutrophils and assess the potential of defensins to control infections caused by organisms that replicate within macrophages. The work with murine neutrophils, whose content of endogenous antimicrobial polypeptides differs greatly from those of humans, will provide essential information relevant to the interpretation of murine models of human infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI022839-12
Application #
2003358
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1985-12-01
Project End
1997-11-30
Budget Start
1996-12-01
Budget End
1997-11-30
Support Year
12
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Doherty, Timothy; Waring, Alan J; Hong, Mei (2006) Peptide-lipid interactions of the beta-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR. Biochim Biophys Acta 1758:1285-91
Tang, Ming; Waring, Alan J; Lehrer, Robert I et al. (2006) Orientation of a beta-hairpin antimicrobial peptide in lipid bilayers from two-dimensional dipolar chemical-shift correlation NMR. Biophys J 90:3616-24
Tang, Ming; Waring, Alan J; Hong, Mei (2005) Intermolecular packing and alignment in an ordered beta-hairpin antimicrobial peptide aggregate from 2D solid-state NMR. J Am Chem Soc 127:13919-27
Mani, Rajeswari; Waring, Alan J; Lehrer, Robert I et al. (2005) Membrane-disruptive abilities of beta-hairpin antimicrobial peptides correlate with conformation and activity: a 31P and 1H NMR study. Biochim Biophys Acta 1716:11-8
Buffy, Jarrod J; Waring, Alan J; Hong, Mei (2005) Determination of peptide oligomerization in lipid bilayers using 19F spin diffusion NMR. J Am Chem Soc 127:4477-83
Buffy, Jarrod J; McCormick, Melissa J; Wi, Sungsool et al. (2004) Solid-state NMR investigation of the selective perturbation of lipid bilayers by the cyclic antimicrobial peptide RTD-1. Biochemistry 43:9800-12
Mani, Rajeswari; Buffy, Jarrod J; Waring, Alan J et al. (2004) Solid-state NMR investigation of the selective disruption of lipid membranes by protegrin-1. Biochemistry 43:13839-48
Owen, S M; Rudolph, D; Wang, W et al. (2004) A theta-defensin composed exclusively of D-amino acids is active against HIV-1. J Pept Res 63:469-76
Wang, Wei; Owen, Sherry M; Rudolph, Donna L et al. (2004) Activity of alpha- and theta-defensins against primary isolates of HIV-1. J Immunol 173:515-20
Cole, Alexander M; Lehrer, Robert I (2003) Minidefensins: antimicrobial peptides with activity against HIV-1. Curr Pharm Des 9:1463-73

Showing the most recent 10 out of 95 publications